We have already demonstrated that the engineered reversal of the β-oxidation cycle can be used to generate straight-chain aliphatic carboxylic acids and n-alcohols with side chains of different lengths and functionalities (61/440,192 and PCT/US12/24051, filed Feb. 7, 2012 and both incorporated by reference herein in their entireties). In all cases the synthesized molecules were primary n-alcohols or carboxylic acids with a methyl group at the omega end. The present invention continues the research developed in the reverse β-oxidation application, and allows further diversification of products.
To summarize the prior ground breaking work, the methodology used to drive the reversed β-oxidation cycle involved the following three steps: 1) functionally expressing the β-oxidation cycle enzymes in the absence of its naturally inducing substrates (i.e. absence of fatty acids) and presence of a non-fatty acid carbon source (e.g. presence of glucose); 2) driving the β-oxidation cycle in the reverse/biosynthetic direction (as opposed to its natural catabolic/degradative direction); and 3) expressing termination enzymes that act on the appropriate intermediate of the β-oxidation cycle to make desired products.
In more detail, the recombinant engineering was:
1) Express the β-Oxidation Cycle in the Absence of its Naturally Inducing Substrates (i.e. Absence of Fatty Acids) and Presence of a Non-Fatty Acid Carbon Source (e.g. Presence of Glucose):
In order to express the β-oxidation cycle, i) mutations fadR and atoC(c) enabled the expression of the genes encoding beta oxidation enzymes in the absence of fatty acids; ii) an arcA knockout (ΔarcA) enabled the expression of genes encoding beta oxidation cycle enzymes/proteins under anaerobic/microaerobic conditions (microaerobic/anaerobic conditions are used in the production of fuels and chemicals but lead to repression of beta oxidation genes by ArcA); and iii) replacement of native cyclic AMP receptor protein (crp) with a cAMP-independent mutant (crp*) enabled the expression of genes encoding beta oxidation cycle enzymes/proteins in the presence of a catabolite-repressing carbon source such as glucose (glucose is the most widely used carbon source in fermentation processes and represses the beta oxidation genes).
2) Driving the Beta Oxidation Cycle in the Reverse/Biosynthetic Direction (as Opposed to its Natural Catabolic/Degradative Direction).
In addition to functionally expressing the β-oxidation cycle, reverse operation of this pathway was accomplished by driving acetyl-CoA and its precursors towards the beta oxidation cycle and preventing A-coA use elsewhere. Specifically, iv) the use of microaerobic/anaerobic conditions minimized the metabolism of acetyl-CoA through the TCA cycle and made acetyl-CoA available to drive the beta oxidation cycle in the reverse/biosynthetic direction; v) pta (or ackA or both), poxB, adhE, yqhD, and eutE knockouts reduced the synthesis of acetate (Δpta or ΔackA and poxB) and ethanol (ΔadhE, ΔyqhD, and ΔeutE) from acetyl-CoA and therefore make acetyl-CoA available to drive the beta oxidation cycle in the reverse/biosynthetic direction; vi) overexpression of thiolases, the first step in the reversal of the beta oxidation cycle, enabled the channeling of acetyl-CoA into this pathway and hence its operation in the reverse direction; vii) ldhA, mgsA, and frdA knockouts reduced the synthesis of lactate (ΔldhA and ΔmgsA) and succinate (ΔfrdA) from pyruvate and phosphoenolpyruvate, respectively, making more phosphoenolpyruvate and pyruvate available for the synthesis acetyl-CoA and therefore making acetyl-CoA available to drive the beta oxidation cycle in the reverse/biosynthetic direction; viii) overexpression of pyruvate:flavodoxin oxidoreductase (ydbK) and acyl-CoA dehydrogenase (ydiO and ydiQRST) enables the coupling of pyruvate oxidation (pyruvate→acetyl-CoA+CO2+Fdred) and trans-Δ2-enoyl-CoA reduction (trans-Δ2-enoyl-CoA+Fdred→acyl-CoA) and hence drive the beta oxidation in the reverse direction.
3) Conversion of CoA Thioester Intermediates to the Desired End Products.
Several termination enzymes that pull reaction intermediates out of the reverse β-oxidation cycle and produce the desired end product were described:
i) CoA thioester hydrolases/thioesterases, or acyl-CoA:acetyl-CoA transferases, or phosphotransacylases and carboxylate kinases for carboxylic acids (i.e. short, medium, and long-chain monocarboxylic acids, β-keto acids, β-hydroxy acids, trans-Δ2-fatty acids),
ii) alcohol-forming CoA thioester reductases for alcohols (i.e. short, medium, and long-chain n-alcohols, β-keto alcohols, 1,3-diols, trans-Δ2-alcohols),
iii) aldehyde-forming CoA thioester reductases and alcohol dehydrogenases which together form alcohols (i.e. short, medium, and long-chain n-alcohols, β-keto alcohols, 1,3-diols, trans-Δ2-alcohols),
iv) aldehyde-forming CoA thioester reductases and aldehyde decarbonylases (which together form alkanes or terminal alkenes of different chain lengths), and
v) olefin-forming enzymes (which together form aliphatic internal alkenes or terminal alkenes or trienes or alkenols).
One or more of these termination enzymes can be overexpressed, as needed depending on the desired end product.
4. Regulation of Product Chain Length.
The chain length of thioester intermediates determines the length of end products, and was controlled by using appropriate termination enzymes with the desired chain-length specificity. Additionally, chain elongation can be inhibited or promoted by reducing or increasing the activity of thiolases with the desired chain-length specificity. These two methods can be used together or independently. For example:
i) knockout of fadA, fadI, and paaJ to avoid chain elongation beyond 1-to-2 turns of the cycle (generates 4- & 6-carbon intermediates and products, or 5- & 7-carbon intermediates and products, depending on the use of acetyl-CoA or propionyl-CoA as primer/starter molecule) and overexpression of the short-chain thiolases yqeF or atoB or short chains alcohol dehydrogenases such as fucO or yqhD;
ii) overexpression of fadB, fadI, and paaJ to promote chain elongation and overexpression of long-chain thiolases tesA, tesB, fadM, ybgC or yciA or long chain alcohol dehydrogenases such as ucpA, ybbO, yiaY, betA, ybdH or eutG.
The term “appropriate” is used herein to refer to an enzyme with the required specificity toward a given intermediate (i.e. acyl-CoA, enoyl-CoA, hydroxyacyl-CoA, and ketoacyl-CoA) of a specific chain length. Please note that the chain length of the thioester intermediates can be controlled by manipulating thiolases (as described above), and hence only thioesters of the desired chain length will be available to the termination enzymes.
We have now modified the above work to make a better platform, which also allows the initiating chemical to include many more primers than just acetyl-coA or propionyl co-A, as well as using an appropriate termination enzyme at step 3 to produce many more additional chemicals.